Comparative resistomics analysis of multidrug‐resistant Chryseobacteria

Abstract Chryseobacteria consists of important human pathogens that can cause a myriad of nosocomial infections. We isolated four multidrug‐resistant Chryseobacterium bacteria from activated sludge collected at domestic wastewater treatment facilities in the New York Metropolitan area. Their genomes were sequenced with Nanopore technology and used for a comprehensive resistomics comparison with 211 Chryseobacterium genomes available in the public databases. A majority of Chryseobacteria harbor 3 or more antibiotic resistance genes (ARGs) with the potential to confer resistance to at least two types of commonly prescribed antimicrobials. The most abundant ARGs, including β‐lactam class A (blaCGA‐1 and blaCIA) and class B (blaCGB‐1 and blaIND) and aminoglycoside (ranA and ranB), are considered potentially intrinsic in Chryseobacteria. Notably, we reported a new resistance cluster consisting of a chloramphenicol acetyltransferase gene catB11, a tetracycline resistance gene tetX, and two mobile genetic elements (MGEs), IS91 family transposase and XerD recombinase. Both catB11 and tetX are statistically enriched in clinical isolates as compared to those with environmental origins. In addition, two other ARGs encoding aminoglycoside adenylyltransferase (aadS) and the small multidrug resistance pump (abeS), respectively, are found co‐located with MGEs encoding recombinases (e.g., RecA and XerD) or transposases, suggesting their high transmissibility among Chryseobacteria and across the Bacteroidota phylum, particularly those with high pathogenicity. High resistance to different classes of β‐lactam, as well as other commonly used antimicrobials (i.e., kanamycin, gentamicin, and chloramphenicol), was confirmed and assessed using our isolates to determine their minimum inhibitory concentrations. Collectively, though the majority of ARGs in Chryseobacteria are intrinsic, the discovery of a new resistance cluster and the co‐existence of several ARGs and MGEs corroborate interspecies and intergenera transfer, which may accelerate their dissemination in clinical environments and complicate efforts to combat bacterial infections.


INTRODUCTION
We are facing an unprecedented threat of antimicrobial resistance (AMR) to our health.It was estimated that 1.27 million deaths in 2019 were directly due to AMRrelated infections across the world (Murray et al., 2022).If the situation is left unsolved, AMR can cause as many as 10 million annual deaths, making it the top 1 killer by 2050 (O'Neill, 2016).Even worse, pathogenic bacteria can evolve from environmental species, particularly those that intrinsically confer resistance to clinical antimicrobials (Lopeman et al., 2019).
They can also acquire new resistance via horizontal gene transfer (HGT), particularly under stress in clinical and other environments with high and frequent antimicrobial exposure (Lerminiaux & Cameron, 2019).
Members of the bacterial genus Chryseobacterium are among such human pathogens that are causal for a myriad of nosocomial infections, including pneumonia, bacteremia, biliary tract, and intra-abdominal infections (Kirby et al., 2004).Increasing infection cases caused by Chryseobacteria have not only been observed in critically ill patients in intensive care units but also in people infected by community-acquired Chryseobacteria.Particularly, infection caused by Chryseobacterium indologenes has remained a critical concern in Taiwan since 1992 (Hsueh et al., 1997).During 1992-1995, an epidemicity of C. indologenes led to 14% death in people who were infected by this pathogen (Hsueh et al., 1997).Other troublesome Chryseobacterium species include Chryseobacterium gleum, Chryseobacterium hominis, Chryseobacterium arthrosphaerae, and Chryseobacterium oranimense.The consistent increase of these pathogenic species in the genus of Chryseobacterium is evident in Africa, Asia, Europe, and America (Kirby et al., 2004;Lin et al., 2019).
Despite being clinic-relevant, Chryseobacteria are also widespread in the environment and can be found in soil, plants, freshwater, municipal wastewater, and drinking water, and a number of these species are known as opportunistic human pathogens (Vandamme et al., 1994).Many environmental and clinical Chryseobacterium species were reported with resistance to antimicrobials and disinfectants, biofilm-forming capacity, and metabolic versatility, promoting them to survive in diverse environments and colonize and infect humans (Dijkshoorn et al., 2007).Some Chryseobacteria are reported to confer resistance to commonly used antimicrobials, particularly those for treating Gram-negative bacteria, such as carbapenems, colistin, tigecycline, and other "last-resort" antimicrobials (Kirby et al., 2004;Mwanza et al., 2022;Zhang et al., 2020).For instance, C. indologenes is resistant to a broad spectrum of β-lactams, including amoxicillin, cephalosporins, and carbapenems due to the expression of Class A (e.g., blaCIA) and/or Class B (e. g., blaIND) β-lactamases (Chen et al., 2013).Furthermore, resistance to environmental stresses (e. g., disinfectants and heavy metals) was also reported in some Chryseobacterium species detected in chlorinated hospital water, downstream of France and Belgium's wastewater treatment facilities, and uraniumcontaminated soil in Northeast India (Arouna et al., 2017;Garcia-Armisen et al., 2011;Khare et al., 2020).Even worse, a recent study isolated 13 different Chryseobacterium strains from food, soil, plant rhizosphere and phyllosphere, and surface water, and these isolates are putative human pathogens considering their ability to express virulence enzymes, such as αor β-haemolyses, proteases, lipases, and DNases (Mwanza et al., 2022).These characteristics of multidrug-resistant Chryseobacteria revealed the potential as a significant and growing threat to global health (Dijkshoorn et al., 2007).
Response to the challenge of multidrug-resistant Chryseobacteria requires a comprehensive understanding of AMR mechanisms and evolutionary traits, which will be beneficial to stimulate the development of appropriate therapeutic approaches.Some recent studies have highlighted the genotypes and phenotypes related to resistance and virulence of Chryseobacterium isolates (Kirby et al., 2004;Mwanza et al., 2022;Victor et al., 2022;Zhang et al., 2020).For example, Mwanza et al. investigated the pathogenicity potential of 37 Chryseobacterium strains isolated from clinical, fish, food, and environmental sources (Mwanza et al., 2022).Another study has centred on virulence factors and secondary metabolites of 73 Chryseobacterium genomes without considering their isolation sources (Victor et al., 2022).However, our current knowledge regarding resistomes and genetic factors that may promote the dissemination of AMR in Chryseobacteria between environmental and clinical settings remains fragmentary.
In this study, association among key antimicrobial resistance genes (ARGs), virulence factors, and other functional genes were untangled in a systematic fashion, engaging the comprehensive comparison of 215 Chryseobacterium genomes from three source categories (i.e., environmental, animal, and clinical settings) available in public databases, as well as those isolated in our lab from activated sludge samples collected at three municipal wastewater treatment plants (WWTPs) in the New York metropolitan area.AMR in these isolates were further validated by assessing their minimum inhibitory concentrations (MICs) to major antimicrobials that are common for clinical use.Our central hypothesis is that the multidrug resistance of Chryseobacteria is dominantly intrinsic, promoting its viability in diverse environments.They can also acquire and disseminate AMR via HGT with transmission potential to human pathogens of close phylogenies, such as Elizabethkingia meningoseptica and Myroides odoratimimus, underscoring their impacts in environmental and human resistomes.

Isolation of multidrug-resistant Chryseobacteria
Activated sludge samples were collected from aeration tanks of three WWTPs in the New York metropolitan area (i.e., L, P, and R sites) between June and October 2019.These WWTPs were selected as they are representative of WWTPs that are of different sizes and wastewater input.These WWTPs serve residential populations in the range from 6.0 Â 10 4 to 1.4 Â 10 6 , as well as a diversity of domestic industries.Ten-time dilution of activated sludge samples was spread onto R2A agar plates amended with 100 mg/L ampicillin, 50 mg/L kanamycin, 20 mg/L tetracycline, and 50 mg/L sulfamethoxazole.After incubation for 2 days at 37 C, multidrug-resistant colonies were formed on plates.Even though seeded with activated sludge from different WWTPs, a majority of these colonies appeared yellow in colour.Based on the 16S rRNA sequencing, four of these yellow colonies were identified as members of the genus Chryseobacterium.This was in agreement with the ability of Chryseobacteria to produce flexirubintype pigments, which impart yellow-orange color colonies.The resistance to these four antibiotics aforementioned for isolation was further validated for these four Chryseobacteria in batch cultures in R2A broth dosed at the same antibiotic concentrations.The genomic DNA of these isolates were extracted for taxonomy identification and whole-genome sequencing.They were also tested to determine their MICs to different antibiotics and susceptibility to UV and chlorine disinfection, as detailed below.

Nanopore sequencing and de novo assembly
High molecular-weight DNA of four sludge-derived Chryseobacterium isolates (P3, R7, L8 and N14) was extracted using the Quick-DNA HMW Magbead kit (Zymo Research, USA), followed by the purification using AMPure XP beads (Beckman Coulter).DNA concentration and quality were examined using the Spec-traMax Plus 384 Microplate Reader equipped with a SpectraDrop Micro-volume Microplate (Molecular Device, CA).DNA samples with OD 260/280 of >1.8 and OD 260/230 of >1.9 were selected for library preparation.Whole-genome sequencing of these isolates was prepared with the SQK-LSK109 1D ligation genomic DNA kit (Oxford Nanopore, UK) following the instruction manual.Long-read sequencing was performed using an Oxford Nanopore MinION flow cell (R9.4.1).Sequencing data acquisition was processed using the MinKNOW software without live base calling.
Raw reads generated from the MinION sequencing were base-called using Guppy, and the passed reads subsequently underwent adapter and barcode trimming using Filtlong v.0.2.0.Quality of the whole-genome sequencing was accessed using NanoPlot v.1.30.1.High-quality reads (base call accuracy >92%) were used for de novo assembly using Flye v.2.8.3 (default settings, except-plasmids).The assembly contigs were subjected to one round of polishing by Racon v.1.4.10 (default setting, except m 8-x-6-g-8-w 500) followed by three rounds of polishing by Medaka v.1.2.3 (Sereika et al., 2022).The quality of the genome before and after polishing was analysed using CheckM (Parks et al., 2015) and shown in Table S1

Collection of Chryseobacterium genomes and quality control
We identified 254 Chryseobacterium's genomes available in the databases of the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) and JGI that includes four genomes of our isolates aforementioned.Information on genome ID, taxonomy, and isolation source is summarized in Data S1.Quality control of these retrieved bacterial genomes was further processed to ensure high quality and minimal bias by following three procedures.First, only isolates with known isolation sources were selected for further investigation.Second, only genomes with at least 95% completeness and less than 5% contamination were used after the examination by CheckM v1.2.1 (Parks et al., 2015).Third, to remove the redundant genomes, FastANI v1.3.2 (Jain et al., 2018) was employed to calculate the whole-genome average nucleotide identity (ANI) and percentage of orthologous matches.The genomes were considered duplicates when the ANI value was >99.995% and the matching percentage was >90% (Levy et al., 2018).Through these three procedures, 215 high-quality and non-redundant genomes of Chryseobacterium isolates were obtained, including 128, 34, and 53 strains isolated from environment, animal, and clinical settings, respectively.

Bioinformatics analysis
For ARG annotation, two approaches were used: (1) the amino acid sequences from Chryseobacterium genomes were searched against the Comprehensive Antibiotic Resistance Database (CARD) (Alcock et al., 2020) and ARG-ANNOT (Gupta et al., 2014) using Blastp with identity and query coverage thresholds of 50% and 50%, respectively; (2) AMRFinder v.3.8.4 (Feldgarden et al., 2019) was used to screen ARGs in the isolate genomes with the same thresholds.Redundant ARGs were manually eliminated from the combined data set.Orthologs of protein-coding sequences were screened using Proteinortho v.6.0.23 with default parameters (Lechner et al., 2011), among which genes that appeared in all 215 bacterial isolates were identified as the core genes of the genus Chryseobacterium as listed in Data S2 (Levy et al., 2018).KEGG Orthology IMG database of core genes was subsequently used to construct KEGG pathways using the KEGG mapper (https://www.genome.jp/kegg/mapper/).
G + C contents and mobile genetic elements (MGEs) within 10-kpb flanking regions of ARGs were determined to identify if certain ARGs are intrinsic.Theoretically, intrinsic ARGs have (1) similar G + C contents to those of core genes detected in their hosts, (2) are chromosomally encoded and (3) are non-mobilized genes.An ARG is considered mobile if one or more MGEs are located within 10 kbp of its flanking region or it is located on a plasmid.The G + C contents of ARGs and core genes were calculated using the bioawk toolkit.MGEs were determined by a string that matches one of the following keywords in the description of genes: integron, integrase, relaxase, type IV coupling, type IV secretory, replication initiator, transposase, transposon, conjugation, conjugative, recombinase, conjugal, mobilization, recombination, and excisionase (Li et al., 2017).The GFF files of IMG and NCBI isolates were converted to BED files, which were further used to search for 10-kbp-flanking regions of ARGs by the Bedtools window (v.2.26.0).In addition, to investigate if ARG located on a plasmid, ARG-containing contigs were searched (1) against the NCBI plasmid database PLSDB (version 2020_06_23) with a minimum similarity of ≥95% and a query coverage of ≥60%, and (2) to analyze if plasmid related genes are present using a keyword of 'plasmid' in the description of genes.

Statistical analysis
The distribution of ARGs, virulent factors, and ortholog genes across the origins (i.e., the environment, animals, and clinical setting) was statistically compared using Fisher's exact test based on the presence or absence of a specific gene in bacterial genomes (Xia et al., 2018).FDR-corrected p values according to Benjamini-Hochberg (a false discovery rate multiple test correction) (Benjamini & Hochberg, 1995) were subsequently estimated.Significant enrichment of certain genes in origin was defined when the adjusted p value was >0.001 compared to another origin.Fisher's exact test was performed using the MASS package within R v4.1.2(R Core Team, 2013).
Correlation of Chryseobacteria isolated from different origins based on their resistome was performed using Bray Curtis distance within QIIME2 (Bolyen et al., 2019;Bray & Curtis, 1957).Pairwise permutational multivariate analysis of variance (pairwise per-MANOVA) was applied to evaluate the significance of differences in Chryseobacterium isolates between each pair of origins using the adonis function (Anderson, 2001).FDR-corrected p values were estimated for pairwise perMANOVA tests.

Antimicrobial susceptibility test
MIC test of our four isolates against ampicillin, cefotaxime, imipenem, kanamycin, gentamycin, chloramphenicol, tetracycline, and rifamycin were determined using broth micro-dilution technique according to CLSI recommendations (Patel et al., 2015).Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for the quality control for the MIC test.

RESULTS
A majority of ARGs detected in 215 Chryseobacterium isolates are potentially intrinsic Chryseobacteria were identified to carry a wide spectrum of ARGs based on the investigation of 215 Chryseobacterium genomes available in the databases of JGI and NCBI.As shown in Figure 1, a total of 82 ARG subtypes belonging to 13 ARG types were detected.Notably, almost all isolates ($98%) contained 3 or more ARGs and confer resistance to at least two different types of antimicrobials.Particularly, an environmental isolate Chryseobacterium sp.POL2 (IMG genome ID of 2,888,279,485) harbors as many as 19 ARGs in its genome.
Approximately 78% of total key ARGs, which include ranA, ranB, blaCIA variants, blaCGA, blaCGB, blaIND variants, blaMYO, abes, rosA, and arr variants, were potentially intrinsic in Chryseobacteria, since (1) none of ARGs are located on plasmids as no ARGs carried contigs were identified as plasmids based on the database analysis, (2) their G + C contents were in a similar range to those of core genes that were shared by all 215 Chryseobacteria genomes (35.9%-40.1% vs. 37.6%) and (3) less than 20% were putatively mobile (Table 1).It is important to note that approximately 50% of Chryseobacterium isolates carry both Class A (i.e., blaCIA variants or blaCGA) and Class B (blaCGB, blaIND variants or blaMYO) β-lactamase genes, accounting for 45%, 50%, and 62% of bacteria with origins from the environment, animal, and clinical settings, respectively.In addition, ranA and ranB, which encode an ABC-type efflux pump system to exclude aminoglycoside from the bacterial cells, were detected in all 215 Chryseobacteria genomes.Another important intrinsic gene is rosA, which was demonstrated as a part of the efflux pump system (rosAB), enabling resistance to cationic peptide antibiotics in Yersinia or other bacteria (Bengoechea & Skurnik, 2000).

Six ARGs enriched in clinical Chryseobacterium isolates
Pairwise perMANOVA test revealed a significant enrichment of several Chryseobacterium-carrying ARGs in clinical isolates as compared to environmental or animal isolates and (FDR-corrected p = 0.003), while no significant difference was observed between environmental and animal isolates (FDR-corrected p = 0.280).Approximately 6 of 82 ARG subtypes, including blaCGA-1, blaCGA-4, blaIND-2, catB11, tetX, and tetX4, were significantly different across the origins (Figure 2).Particularly, catB11 and tetX were found to be significantly enriched by approximately one order of magnitude in clinical isolates as compared to those with environmental origins where exposure to anthropogenic interferences is less in general (Fisher's exact test, adjusted p < 0.001) (Figure S1).Specifically, 54.2% of clinical isolates contain catB11, while only 8.3% of environmental isolates carry the same gene.Similarly, 41% of clinical isolates contain tetX, while only 4.5% of environmental isolates carry this ARG.Enrichment of catB11 at a factor of 8.83 was also evident in clinical isolates in comparison to animal isolates.54.2% of clinical isolates contain catB11, whereas it is only found in 10.5% of animal isolates.

Three ARGs with transmission potentials within the Bacteroidota phylum
Though the majority of ARGs are putatively intrinsic, three ARGs (Table 2) harbored by Chryseobacteria were identified with high potential to be transmissible among species within the Bacteroidota phylum, including catB11, tetX, and aadS, which confer resistance to chloramphenicol, tetracycline, and aminoglycosides, respectively.
Genome mining of catB11 and tetX in 215 Chryseobacterium genomes and other bacterial genera from the NCBI non-redundant (nr) database revealed a resistance cluster consisting of both catB11 and tetX genes in 9 Chryseobacterium isolates from different sources with high similarity of >90% (Table S2).As shown in Figure 3, this resistance cluster is often located next to the IS91 family transposase and tyrosine-type recombinase XerD in the Chryseobacterium genomes.The co-occurrence of catB11 and tetX was also identified in other species, such as Empedobacter brevis strain SE1-3 and Elizabethkingia anophelis strain EA1 (Figure 3).However, this co-existence is not common in other Bacteroidota, even though the tetX gene (WP_008651082.1) is frequently detected in 15 genera belonging to the Bacteroidota phylum, as well as the Aliarcobacter genus of the Proteobacteria phylum.Meanwhile, catB11 (WP_035589996.1) is less frequent and detected in <5 genera of the Bacteroidota phylum.
Approximately 35% of 134 aadS genes co-existed with MGEs encoding recombinases (e.g., RecA and XerD) or transposases.As depicted in Figure 4A, the environmental isolate Chryseobacterium sp.POL2 carries an aadS gene that is 100.0%identical to that identified in the pathogen M. odoratimimus strain PR63039.The aadS gene in POL2 is also located in a 2.7-kbp flanking region that overlaps with the genome  of PR63039, implying these sequences between these two bacteria may share an identical origin.Similarly, the genome of an animal isolates Chryseobacterium sp.SNU WT5 shared >99.4% identity to both aadS and the 8.6-kbp region in the pathogen E. anophelis NUHP1 (Figure 4B).AadS genes (NCBI accession number of AAA27459.1)were not only identified in Chryseobacteria, but also in 13 other genera that belong to the Bacteroidota phylum according to NCBI identical protein groups.High similarity (>98%) of aadS genes and their flanking regions between Chryseobacteria and other species within the Bacteroidota phylum (e.g., Myroides, Elizabethkingia, Bacteroides and Riemerella) is detailed in Table 3.These results suggest HGT of aadS-containing fragments among Bacteroidota regardless of their pathogenicity (Forsberg et al., 2012).

Antibiotic
Collectively, the co-occurrence of three ARGs mentioned above (i.e., catB11, tetX, and aadS) and MGEs (e.g., recombinase and transposase genes) and their high identity among Chryseobacteria and other members of the Bacteroidota phylum underline their critical role in disseminating AMR to aminoglycosides, chloramphenicol, and tetracycline.

Mobility of a multidrug resistance gene abeS associated with a RayT transposase gene
A putative insertion of the transposase gene RayT to 10-kbp-flanking regions of abeS was observed in many Chryseobacteria of clinical origins with previous anthropogenic disturbance (Figure 5).AbeS encodes a small multidrug resistance pump that enables bacteria to confer resistance to aminocoumarins and macrolides and this ARG was detected in 139 Chryseobacterium isolates (Srinivasan et al., 2009).The colocation of both abeS and RayT was found in 15 clinical and 2 environmental isolates (Table S3).Fisher's exact test revealed a significantly higher abundance of this abeS-RayT cluster in clinical isolates as compared to those detected in environmental isolates (FDR p < 0.05).Chryseobacteria carrying the abeS-RayT cluster were isolated from clinical settings, activated sludge, and Populus root rhizosphere, which are often related to antimicrobial exposure history (Table S3) (Chen et al., 2019;Ju et al., 2019).Meanwhile, Chryseobacteria without the abeS-RayT cluster were often associated with environments with little anthropogenic activities, such as glaciers, soil and leaf.
As shown in Figure 5, in two C. indologenes isolates, the RayT transposase is located within 10 kbp at the flanking region of abeS.In contrast, this MGE is >1800 kbp away from the abeS gene in the chromosome of Chryseobacterium glacier species isolated from a glacier.Furthermore, RayT transposase genes located within the 10-kbp flanking region of abeS exhibited much higher similarities (>74.4% for amino acid sequences) than other RayT transposase genes identified in Chryseobacterium genomes.Phylogenetic analysis revealed that these RayT genes can cluster into an ortholog, suggesting they might have derived from a common ancestor (Lechner et al., 2011).Genome mining also revealed the abeS and its cluster with the transposase gene RayT shared <81.0%amino acid sequence similarity with bacterial genomes other than the Chryseobacterium species, suggesting this abeS-RayT cluster might have been recently formed within the genus Chryseobacterium via interpopulation transmissions (Jiang et al., 2017).

Majority of virulence factors independent of the origins
In the 215 Chryseobacterium genomes, a total of 4677 genes were predicted and classified into 83 virulence factors.A majority of virulent factors were clustered into groups of adherence, immune response, mobility, nutritional/metabolic factor, and stress survival (Figure S2).Similar to the ARG profile, the majority (>93%) of predicted virulence factors were not significantly enriched in the clinical isolates as compared with other isolates from animals and environment (Table S4).Furthermore, no distinctive clusters were identified among Chryseobacterium isolates from environmental, animal, F I G U R E 2 Significant differences in key ARGs between different sources.The heatmap shows the level of enrichment based on Fisher's exact test with adjusted p < 0.001.
F I G U R E 3 (A) Comparative analysis between the catB-tetX-flanking regions in Chryseobacterium genomes and those from the genomes of other species in the Bacteroidota phylum.Shades show conserved regions of higher than 94% similarity in nucleotide sequences.(B) Two models of tetX-catB11 resistance clusters.and clinical sources regarding the phylogenetic analysis based on virulence factor profile (Figure S2), corroborating the close relevance of environmental, animal, and clinical isolates regarding their pathogenicity.
The attachment of Chryseobacteria to host cells is the first critical step for infection and is associated with adherence genes, such as Hsp60, EF-Tu, and IlpA.It is important to note that Hsp60 was found in all F I G U R E 4 Comparative analysis between the aadS-flanking regions in Chryseobacterium genomes and those from the genomes of other species in the Bacteroidota phylum.Shades show conserved regions of higher than 98% similarity in nucleotide sequences.

ENVIRONMENTAL MICROBIOLOGY REPORTS
215 Chryseobacterium genomes.Following the primary surface adhesion, the growth of attached bacteria such as Chryseobacteria can be mediated by factors relevant to immune response, mobility (e.g., flmH), nutritional/metabolic traits (e.g., mgtB, panD).In Chryseobacteria, an immune response is predominant by the expression of capsule and formation of lipopolysaccharide (LPS) and lipooligosaccharide (LOS).Several highly abundant genes involved in the capsule biosynthesis pathway include cap8E, cap8G, and tviB.
In addition, acpXL, galE, rffG, wbpD, hisF2, lpxA, lpxD, rfbC, wbtL, and wbtF, which are involved in the biosynthesis of LPS/LOS, were frequently detected among Chrseobacterium species.Several virulence factors associated with stress survival enabling bacterial pathogens to persist in host cells were also detected in Chryseobacteria, including ClpP, UreB, UreG, KatG, KatAB, KatA, and icl.ClpP was detected in all 215 Chryseobacterium isolates.Detailed discussions regarding these virulent factors are provided in the Supporting Information.

DISCUSSION
This study was initiated by our observation that Chryseobacteria were dominant multidrug-resistant bacteria screened by the plating method among different activated sludge samples collected in the New York Metropolitan area.Their resistance to commonly prescribed antimicrobials was further confirmed by MIC assays and the detection of corresponding ARGs in the genomes of these four isolates obtained in our lab.This consensus among our isolates stimulated our interest beyond these isolates to deepen our understanding of resistomes of Chryseobacteria at the genus level.
To date, though many emerging antimicrobial-resistant pathogens have been identified (Sanz-García et al., 2021;Vouga & Greub, 2016), only a few studies have performed a thorough comparison between clinical and non-clinical species, such as Mycobacterium abscessus (Davidson et al., 2022), P. aeruginosa (Rossi et al., 2021), and Herbaspirillum seropedicae (Faoro et al., 2019).Considering the multidrug resistance of Chryseobacteria and potential pathogenicity, we conducted a comparative genomics analysis based on genomes available at NCBI and other databases in response to their origins categorized as environmental, animal, and clinical.It is notable that approximately 98% of 215 Chryseobacterium genomes contained more than three ARGs and the majority of them were intrinsic (Figure 1; Table 1).Intrinsic ARGs are recognized to be stably maintained in Chryseobacteria so they can retain the resistance with less regard to the change in the environment and growth condition (Mançano et al., 2020).Up to 50% of Chryseobacterium isolates carry both Class A and B β-lactamase genes, such as penams, cephalosporins, and carbapenems, and even the last-resort antibiotics imipenem and meropenem.This trait probably enabled Chyseobacterium species to confer resistance to a broad spectrum of βlactams.This is in good agreement with previous epidemiological analyses of Chryseobacteria isolated from Asian, European, and American hospitals in different investigation periods during 1994-2019 (Chen et al., 2013;Kirby et al., 2004;Zhang et al., 2020).For instance, >90% of 215 clinical C. indologenes strains conferred resistance to all tested β-lactams, including the last-resort antibiotics imipenem and meropenem (Chen et al., 2013).In addition, our discovery of the frequent existence of rosA in Chryseobacterium genomes may explain the resistance to colistin reported previously, as this ARG encodes a cationic antimicrobial peptide that is important for cystic fibrosis treatments (Sharma et al., 2015).
Phenotype analysis confirmed the resistance for all four sludge-derived Chryseobacterium strains isolated in our lab to commonly used antibiotics except rifamycin despite the presence of arr and arr-5 genes (Table 2).The presence of cryptic ARGs such as arr and arr-5 genes is common in bacteria.For example, 16% of Acinetobacter baumannii strains contained blaOXA-23 but remained sensitive to imipenem (Deekshit & Srikumar, 2022).However, cryptic ARGs can be activated through various mechanisms, such as (1) mutagenesis triggered by the exposure to the corresponding antibiotics, (2) HGT to other species that harbour a positive transcriptional regulator or nondefective promoter of acquired ARGs and (3) change the growth medium (Deekshit & Srikumar, 2022;Stasiak et al., 2021).
Some non-intrinsic ARGs showed a significant enrichment between clinical and environmental/animal Chryseobacteria species, implying the acquisition of new ARGs to clinical isolates and their potential to transmit these ARGs via HGT.In our study, a resistance cluster of catB11-tetX was significantly enriched in clinical isolates as compared to those with environmental origins (Figure 2).The selection for the catB11-tetX resistance cluster may be due to some long-term use of antimicrobial cocktails with chloramphenicol and tetracycline for bacterial infection in the hospital.For example, six clinical-related Chryseobacterium strains carrying catB11-tetX clusters were isolated during the period of 2004-2019 from seven different hospitals in China.The clinical usage of chloramphenicol and tetracycline in China was estimated at approximately 215 and 1170 tons per year in 2013, respectively (Zhang et al., 2020).Interestingly, several other ARGs are also found within or in proximity to these catB-tetX resistance clusters, including ereD, aadS, aar, MphE, and sul2 (Figure 3A).For instance, ereD was found in four out of nine catB-tetX resistance clusters in Chryseobacteria.The co-existence of catB11, tetX, and other ARGs may promote a greater ecological fitness, thereby increasing the adaptability of bacteria to extensive stresses under clinical conditions.
Furthermore, catB11 and tetX are located in the proximity of recombinase and transposase genes (e. g., IS91 and XerD) (Figure 3B), promoting HGT of these ARGs via mechanisms, such as site-specific recombination and random relocation, concurrently spreading both ARGs and many others (Partridge et al., 2018;Zhang et al., 2015).This resistance cluster of catB11-tetX, along with recombinase and transposase genes, are also identified in other genera within the Bacteroidota phylum (Figure 3), suggesting interspecies and intergenera dissemination.Besides pathogenic Chryseobacterium species, many Bacteroidota species such as E. meningoseptica, Bacteroides fragilis, M. odoratimimus can cause serious infections that are often associated with high mortality rates due to their resistance to many antimicrobials (Wexler, 2007;Zhang et al., 2020).The co-existence of ARGs and associated MGEs in Bacteroidota members calls for attention, considering the increased mobility and associated health impact of these ARGs.Previous studies have revealed a multitude of resistance gene clusters in different bacteria (Partridge et al., 2018).For instance, a cassette consisting of a resistance gene cluster containing disinfectant resistance gene (qacE) and sulfonamide protection gene (sul1) and the Tn402-intI1 hybrid have been identified to be widespread in a wide variety of environmental and pathogenic bacteria in different habitats (Gillings et al., 2015).
The small multidrug resistance pump (abeS) may also be a response to previous anthropogenic interference since the insert of the RayT transposase gene to the flanking regions of abeS significantly occurred at a higher frequency in clinical isolates than those in environmental isolates (Figure 5; Table S3).Multidrug resistance pumps such as abeS are often considered as native elements in bacteria and not commonly detected next to the MGEs such as transposons (Alekshun & Levy, 2007).Recent studies have revealed the persistence of efflux pump systems in bacterial genomes can extend resistance to a multitude of antimicrobials, such as fluoroquinolones, β-lactams, tetracyclines, and phenicols (Alekshun & Levy, 2007;Tikhonova et al., 2011).Therefore, the colocation of abeS and RayT in certain Chryseobacteria species may promote bacterial viability and proficiency when exposed to the stress of a broad spectrum of antimicrobials.
In addition to ARGs, we also found a number of pathogenicity-associated genes potentially encoding adhesion, capsular, LPS/LOS, and virulent extracellular enzymes frequently detected among the Chryseobacterium isolates (Figure S2).Therefore, our study has identified multidrug-resistant Chryseobacteria as potential emerging pathogens with a comprehensive understanding of their resistome and virulence.The discovery of a new resistance cluster tetX-catB11-IS91-XerD and the colocation of abeS and RayT facilitate future investigation of resistance transfer mechanisms, ongoing transmission patterns, and genomic rearrangement events among the members that belong to or are beyond this genus, and subsequently promote the development of new antimicrobial therapy for infectious diseases caused by Chryseobacterium species.Furthermore, we identified prevalent ARGs and virulence factors of the Chryseobacterium genus, such as ranA, ranB, Hsp60, and ClpP, which can be employed to timely track the abundance and dynamics of Chryseobacteria in contamination events and human disease cases, leading to efficient monitoring and mitigation of future outbreaks.In addition, ARGs and virulence factors that were significantly enriched in clinical-related Chryseobacteria (e.g., catB11, tetX, and icl) can be used as biomarkers to track the pathogenic Chryseobacterium populations.Beyond providing an evolutionary perspective for clinically relevant versus other Chryseobacteria, our study also offers a comparative resistomics strategy for investigating the evolution of other human pathogens of emerging concern.

F
I G U R E 1 A phylogenetic tree of 215 Chryseobacterium genomes based on the resistomes.The left column depicts the three sources of Chryseobacterium isolates (i.e., animal, clinical and environment), and the right heat map shows the abundance of antibiotic resistance genes (ARGs) found in the genomes of these isolates.
T A B L E 1 Characteristics of key ARGs of Chryseobacteria about their detection frequency, G + C content, percentage of mobile ARGs, and major MGEs detected within 10 kbp of individual ARGs.
T A B L E 3 AadS-carrying contigs in Chryseobacteria with high similarity to those detected in pathogenic species within the Bacteroidota phylum.

F
I G U R E 5 Potential genome rearrangements in the flanking regions of abeS based on the comparative analysis between the environmental and clinical Chryseobacterium isolates.